Synthesis of estrogens in progenitor cells of adult fish brain: evolutive novelty or exaggeration of a more general mechanism implicating estrogens in neurogenesis?

In contrast to other vertebrates, in which the adult brain shows limited adult neurogenesis, teleost fishes exhibit an unparalleled capacity to generate new neurons as adults, suggesting that their brains present a highly permissive environment for the maintenance and proliferation of adult progenitors. Here, we examine the hypothesis that one of the factors permitting establishment of this favourable environment is estradiol. Indeed, recent data showed that radial glial cells strongly expressed one of two aromatase duplicated genes. Aromatase is the estrogen-synthesizing enzyme and this observation is of great interest, given that radial glial cells are progenitor cells capable of generating new neurons. Given the well-documented roles of estrogens on cell fate, and notably on cell proliferation, these data suggest that estradiol could be involved in maintaining and/or activating these progenitors. Examination of recent data in birds and mammals suggests that the situation in fish could well be an exaggeration of a more general mechanism implicating estrogens in neurogenesis. Indeed, there is accumulating evidence that estrogens are involved in embryonic, adult or reparative neurogenesis in other vertebrates, notably in mammals.

Differential distribution of androgen and estrogen receptors in rat pituitary cell populations separated by centrifugal elutriation.

The distribution of 5 alpha-dihydrotestosterone (DHT)-binding sites within specific pituitary cell populations, as compared with the estradiol (E2)-binding sites distribution, could be estimated after gonadotrope cell enrichment. Centrifugal elutriation allowed the purification of large gonadotropes from 42-day-old male rat pituitaries; this population contained 5.3 +/- 1.0% of the cells, 54 +/- 3% of the LH, and less than 5% of PRL. A gonadotrope-depleted fraction was also defined that contained more than 92% of the total PRL and was thus called the lactotrope population though it still contained small gonadotropes and most somatotropes. DHT and E2 receptors were quantified by multidose saturation analysis. The association constants (Ka) ranged from 0.2-0.5 X 10(9) M-1 both for E2 and DHT independent of the cell population. The binding of DHT was mainly observed in the gonadotropes (50-100 fmol/10(6) cells), while it was very low in the other cells (0.9-3 fmol/10(6) cells). E2-binding sites measured within the same cell populations showed a broader distribution than DHT-binding sites. Whereas the ratio of binding DHT/E2 averaged 0.86 +/- 0.01 in the gonadotropes, it reached only 0.35 +/- 0.07 in the lactotropes. Thus, using enriched cell populations, our results demonstrate for the first time a quantitative selective distribution of androgen-binding sites as compared with the estrogen sites.

Multiple forms of affinity-labeled estrogen receptors in rat distinct pituitary cells.

The presence of multiple monomeric forms has been described for estrogen receptor (ER) in different target tissues. Using [3H]tamoxifen aziridine ([3H]TA) to covalently label ER and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to analyze labeled products, ER forms were investigated in pituitary cytosol and purified nuclei from male rats. ER forms were also compared in cellular extracts from gonadotrope-enriched populations (GP), prepared using the fast method of centrifugal elutriation, and from lactotrope-somatotrope fractions (LSP), obtained by sequential use of both elutriation and Percoll gradient sedimentation. A major labeled protein of 60,000-65,000 mol wt (M(r)) and a minor species of 50,000-55,000 M(r) were found in the pituitary cytosol and nuclear extracts covalently labeled with [3H]TA. The same results were obtained after ER covalent labeling from cellular extracts or intact dispersed cells. In gonadotrope-enriched cell population (greater than or equal to 50% LH-immunoreactive cells), the 65,000 M(r) species is the single unique ER form; in the LSP (80% PRL- and GH-immunoreactive cells), the major TA-labeled species is the 50,000 M(r) form, while the 65,000 M(r) ER is hardly detectable. Thus, the prevalence of 65,000 M(r) protein in the initial cell population can be explained by the higher number of binding sites per gonadotrope than per lactotrope cell. In conclusion, ER heterogeneity is demonstrated in pituitary cell populations. The source of this heterogeneity could be due to 1) different ER mRNAs according to cell type, or 2) a specific posttranslational processing, such as proteolytic activity within lactotrope cells.

Evidence for 5 alpha-androstane-3 beta, 17 beta-diol binding to the estrogen receptor in the cytosol from male rat pituitary.

The binding of [3H]5 alpha-androstane-3 beta, 17 beta-diol (3 beta-Adiol) in the pituitary cytosol of prepubertal male rats was studied. The following criteria indicated that 3 beta-Adiol was bound to specific proteins and, in all probability, to the estrogen receptor. 1) The binding was of relatively high affinity and low capacity. 2) The complex was destroyed by proteolytic enzymes and by heating. 3) It was precipitable with protamine sulfate and 35% ammonium sulfate. 4) In sucrose linear gradients (5-20%), 3 beta-Adiol was bound in the 7S region. The 3-4S 3 beta-Adiol complex, previously found, was due to the dissociation of 3 beta-Adiol from the 7S complex and further association with nonspecific proteins. 5) Bound hormone was only displaced by 3 beta-Adiol itself and estrogens. 6) Translocation of the estrogen receptor from cytosol to nuclei resulted in a parallel decrease in the number of cytosol 3 beta-Adiol-binding sites. It was also shown that 3 beta-Adiol was not bound to contaminating serum proteins, particularly to alpha-fetoprotein. We interpret these results to indicate that in the male rat pituitary, 3 beta-Adiol is bound to the estrogen receptor.

Ontogeny of 5 alpha-androstan-3 beta, 17beta-diol and 17 beta-estradiol binding to cytoplasm and nuclei of the male rat.

The concentrations of estradiol (E2) and 5 alpha-androstane-3 beta, 17 beta-diol (3 beta-Adiol) receptor were quantified in the pituitaries of male rats, aged 15--100 days. The available binding sites were measured directly in pituitary cytosol. E2 receptor levels reached a peak between days 22 and 30 and then declined slightly. 3 beta-Adiol-binding sites increased between days 15 and 37, reached a maximum level at the time of puberty (37--42 days of age), and then declined markedly to become undetectable on day 75. Occupation of cytosol binding sites by endogenous steroid was investigated by means of an exchange assay. No occupation was seen for E2 binding. On the other hand, 3 beta-Adiol-binding sites were partly occupied on day 43 and completely occupied on day 75. The E2 and 3 beta-Adiol association constants remained relatively constant throughout the entire period studied. Total E2- and 3 beta-Adiol-binding sites were measured by an exchange technique in nuclear extracts. The ontogenic patterns of E2- and 3 beta-Adiol-binding sites were similar at all ages. The number of E2- and 3 beta-Adiol-binding sites reached a maximum on days 28--30 and remained constant up to day 75. These data demonstrate the developmental appearance of pituitary E2 and 3 beta-Adiol receptors in the male rat. They suggest two forms of estrogen receptor in pituitary cytosol; one could be involved in the onset of puberty. Moreover, the nuclear ontogenic pattern suggested a single class of binding sites for E2 and 3 beta-Adiol in the pituitary nuclei.

Novel intronic promoter in the rat ER alpha gene responsible for the transient transcription of a variant receptor.

To analyze the molecular origin of an ER variant, the truncated ER product-1, transiently expressed at the proestrus in lactotrope cells, we generated a 2.5-kb sequence of a genomic region upstream and downstream the specific sequence truncated ER product-1. Genomic Southern blot analysis showed that truncated ER product-1 is spliced from a noncoding leader exon localized within the intron 4 of the ER alpha gene. Analysis of the promoter sequence revealed the presence of a major transcriptional start site, a canonical TATA box and putative cis regulatory elements for pituitary specific expression as well as an E-responsive element. In transient transfection, the truncated ER product-1 promoter was transcriptionally the most active in the lactotrope cell lines (MMQ). Analysis of truncated ER product-1 functionality showed that: 1) the protein inhibited ER alpha binding to the E-responsive element in electromobility shift assays, 2) inhibited the E2 binding to ER alpha in binding assays, 3) the truncated ER product-1/ER alpha complex antagonized the transcriptional activity elicited by E2, 4) nuclear localization of green fluorescent protein-ER alpha was altered in Chinese hamster ovary cell lines stably expressing truncated ER product-1. Collectively, these data demonstrated that the protein exerts full dominant negative activity against ER alpha. Moreover, truncated ER product-1/ER alpha complex also repressed the activity of all promoters tested to date, suggesting a general inhibitory effect toward transcription. In conclusion, the data suggest that truncated ER product-1 could regulate estrogen signaling via a specific promoter in lactotrope cells.

Steroid-independent activation of ER by GnRH in gonadotrope pituitary cells.

In the rat pituitary gland the mechanism responsible for ERalpha regulation has not been fully elucidated. Using transient transfection assays in alphaT3-1 cells, a cell line of gonadotrope origin, we show that GnRH stimulates estrogen response element-containing promoters in an estrogen-independent manner. This effect was strictly ER and GnRH receptor dependent, as no activation of the reporter gene was observed in presence of the anti-estrogen ICI 182,780 or a GnRH antagonist. These data suggest that the GnRH-triggered signaling pathway results in 17beta-estradiol-independent trans-activation of the ERalpha in alphaT3-1 cells. Furthermore, an additive activation was achieved when cells were treated with both GnRH and 17beta-estradiol. In primary pituitary cells, GnRH alone (100 nM) did not cause a significant stimulation of reporter gene activity, presumingly due to the low amount of gonadotropes. Interestingly, the combination of 17beta-estradiol and GnRH resulted in a significant increase in ERalpha trans-activation compared with that in cells treated with 17beta-estradiol alone. This enhancement was prevented by ICI 182,780, showing an ERalpha requirement. Moreover, we show that the effects of GnRH on ERalpha transcriptional activity in gonadotrope cell lines are mediated by the PKC/MAPK pathway. In conclusion, our data demonstrate that GnRH is an important signal in the regulation of ERalpha trans-activation in gonadotrope cells.

Peptides co-released with luteinizing hormone by perifused pituitary cell aggregates.

The proteins co-released with gonadotropins were analyzed using perifusion of pituitary cell aggregates from 14-day-old female rats, after a pre-labeling period with [35S]methionine. Radioimmunoassays of hormones and electrophoretic analysis were performed on each 4 min effluent. Gonadotropin-releasing hormone (GnRH) pulses increased significantly (P less than 0.01) the release of several proteins (Mr range from 140,000 to 28,000). The main stimulation appeared for -1, a 87 kDa species, previously characterized as gonadotrope polypeptide 87 (GP87) in monolayer cultures and identified as a secretogranin II (SgII) form; -2, a second species of 80 kDa designated as B2. Secretory patterns of radiolabeled GP87 and B2 paralleled the luteinizing hormone (LH) ones. The release of these species was -1, GnRH dose dependent; -2, monophasic for short pulses but complex when the duration of GnRH pulses increased to 16 min, suggesting different pools of GP87 and B2 as for LH; -3, induced by thyrotropin-releasing hormone (TRH). A slight output was also elicited by corticotropin-releasing factor (CRF) and growth hormone-releasing hormone (GHRH), but this release was partly impaired in the presence of a potent anti-GnRH ([Ac-D-(2)-NAL1,pF-D-Phe2,D-Trp3,D-Arg6]-LAF) suggesting a non-specific effect of these two factors. GP87/SgII thus appeared mainly associated with the release of hormonal glycoproteins. In conclusion, perifusion of pituitary cell aggregates allows a precise minute-to-minute kinetic analysis of the various proteins co-released with hormones. The similar timing in output of LH, GP87 and B2 suggests that these three proteins co-exist in the same secretory granules inside gonadotropes.

The sheep estrogen receptor: cloning and regulation of expression in the hypothalamo-pituitary axis.

We have prepared an ovine pituitary cDNA library, isolated a clone containing the full-coding sequence of estrogen receptor (ER) cDNA, and determined its primary structure. This cDNA encodes a protein of 596 amino acids which shows great homology to other mammalian ER sequences, the highest degree being 95% with the porcine receptor. Northern blot analysis of ovine pituitary RNA revealed a 6.3 kb transcript. This receptor was showed to bind a consensus ERE and to be transcriptionally activated by E2. Studies investigating the pattern of expression of the ovine ER mRNA were also carried out, using the reverse transcription/PCR technique. Expression of ER mRNA was analyzed in ram pituitary and hypothalamus after contrasted light regimen and castration. Results showed that the light regimen had no effect on ER mRNA expression whereas castration induced a slight (approximately 20%) but significant increase of ER mRNA expression at both the hypothalamic (P < 0.05) and pituitary (P < 0.01) levels, indicating a negative regulation of ER gene expression by testicular steroids. Since we have previously shown no variations in ER protein levels after castration, data suggest the activation of a complex pattern including both transcriptional and post-transcriptional regulatory mechanisms in the ram hypothalamo-pituitary axis.

Steroid receptors and regulation of LH and secretogranin II (GP87) secretion in pituitary cell aggregates.

The gonadotrope polypeptide (GP87), a secretogranin II form, has previously been found to be co-released with luteinizing hormone (LH) by rat gonadotrope cells under luteinizing hormone-releasing hormone (LHRH) stimulation. Nothing is hitherto known concerning its function and regulation. In the present paper, the modulatory effects of steroids on GP87 release were investigated using cultures of rat pituitary cell aggregates. As steroid effects are dependent on the presence of receptors, we firstly demonstrated that the steroid receptors are maintained with their typical characteristics in this culture system. In steroid-free medium, the estrogen receptor concentration increased significantly (P < 0.01) within 7 days in culture (646 +/- 122 fmol/mg DNA, n = 3) when compared to the level in freshly dispersed cells (355 +/- 59 fmol/mg DNA, n = 4) suggesting that cells can synthesize the estrogen receptor and that this cell culture system is suitable for studying regulatory effects of steroids. Pituitary cell aggregates from 14 day-old female rats were maintained for 24 h in the absence or presence of 10 nM estradiol (E(2)) or 50 nM 5?-dihydrotestosterone (DHT) and incubated for 2.5 h with [(35)S] methionine. Perifusion was initiated and 4 min pulses of 20 nM LHRH occurred every 1 h. The effects of steroids were investigated on LH, total and labeled GP87. The results indicated that: (1) DHT pretreatment reduced the amplitude of LHRH-stimulated GP87 and LH release responses; (2) by contrast, E(2) pretreatment enhanced the effects of LHRH on total or labeled GP87 release whereas the LH release was not significantly modified; and (3) when E(2) treatment started only 60 min before the first LHRH pulse, no differences between responses of E(2)-treated and control aggregates were observed. The present data show for the first time that sex steroids can modulate the GP87 release through a direct effect on the pituitary. E(2) could either stimulate the GP87 synthesis or increase its intracellular trafficking. That LH and GP87 do not strictly exhibit the same release response further suggests either some heterogeneity of GP87 localization in gonadotrope sub-types or within secretory granules.

Rapid and intensive conversion of 5alpha-androstane-3alpha, 17beta-diol into 5alpha-dihydrotestosterone in the male rat anterior pituitary: in vivo and in vitro studies.

The in vivo and in vitro metabolism of (3H)-5alpha-androstane-3lapha,17beta-diol by the male rat anterior pituitary was studied. A rapid and intensive conversion of 5alpha-androstane-3alpha, 17beta-diol into 5alpha-dihydrotestosterone was demonstrated, since following a 30 min. incubation time, 73% of the recovered radioactivity were constituted by 5alpha-dihydrotestosterone. Studies on the subcellular distribution of steroids showed that 5alpha-dihydrotestosterone was the main steroid recovered except from the 105,000 X g pellet. From in vivo and in vitro experiments it was concluded that the transformation of 5alpha-dihydrotestosterone into 5alpha-androstane-3alpha, 17beta-diol was a reversible process, and that this last steroid could exert its biological action mainly via 5alpha-dihydrotestosterone.

Combretodendron Africanum bark extract as an antifertility agent. I: Estrogenic effects in vivo and LH release by cultured gonadotrope cells.

An aqueous extract from the stem bark of Combretodendron africanum was prepared to investigate its potency as a regulator of fertility. The intraperitoneal LD50 in mice was 95.2 +/- 4.3 mg/kg. Daily i.p. injections of 50 mg/kg promoted a significant increase in the uterine weight of 25 day-old female rats while pituitary weight remained unaffected. Similar injections for 21 days to mature females blocked the estrous cycle in the luteal phase with a mean length of efficiency of 15 days and decreased plasma LH and FSH levels. Regular cycles were restored 10 days following the last injection. C. africanum extract was shown to compete with estradiol and with progesterone on uterine receptors. Consequently, it is thought to contain substances exhibiting estrogenic (and possibly anti-estrogenic) potency. The effects of the extract on LHRH-induced release were investigated on cultured pituitary cells. Although gonadotropin release was amplified, the extract itself appeared as to be a potent secretagogue not requiring LHRH receptors. It follows that the active molecules contained in C. africanum extracts may be different from classical steroid estrogens.

Mechanisms underlying antigonadotropic effects of some traditional plant extracts in pituitary cell culture.

Previous works have demonstrated that stem bark extracts of Cola nitida (Sterculiaceae), Afrormosia laxiflora and Pterocarpus erinaceus (Fabaceae) provoked a blockade of female rat ovulation and estrous cycle by inhibiting pituitary LH release in vivo. In addition, these plant extracts exerted an inhibitory effect on LH release of rat pituitary cells. Therefore, these data could explain inhibitory effects of plant extracts on LH release in vivo. The present study was undertaken to examine the mechanisms by which these plants exert their antigonadotropic activities. So, we studied the biological activities of these plant extracts on pituitary cells in culture. Data show that C. nitida, A. laxiflora and P. erinaceus extracts only inhibit LH release and have no effect on FSH release. In fact, A. laxiflora, P. erinaceus and C. nitida extracts diminish LH release in culture medium without acting on rat pituitary cell content. Plant extracts form complexes with basic glycoproteins (but not with acid glycoproteins) and prevent them from entering the cells.

Long-term castration decreases the androgen but not the estrogen nuclear pituitary receptors in the ram.

The effect of castration on pituitary androgen and estrogen nuclear receptors was examined in eleven 3-year-old 'Préalpes du Sud' rams which were either intact (N = 5) or surgically castrated (N = 6) 11 months before. To avoid a receptor loss, pituitaries were frozen less than 1 min after slaughter. Specific androgen and estrogen bindings were measured upon extracts from purified nuclei. Neither the concentration of estrogen receptors (30.5 +/- 3.0 vs 28.1 +/- 3.0 fmol/mg protein) nor the affinity constant (ka = 1.21 X 10(9) mol-1 in both groups) differed between intact and castrated rams. Conversely, androgen receptor concentrations differed markedly between groups and were found significantly higher in intact rams (11.3 +/- 1.1 fmol/mg protein) than in castrated rams where nuclear receptors were undetectable in 5 out of 6 animals. This result keeps open the possibility that the decrease of LH sensitivity to testosterone negative feedback observed after long-term castration, could be related to the quasi absence of nuclear receptors, at least at the pituitary level.

Interaction of some traditional plant extracts with uterine oestrogen or progestin receptors.

Previous work has demonstrated that stem bark extracts of Combretodendron macrocarpum (Barringtoniaceae), Cola nitida (Sterculiaceae), Afrormosia laxiflora and Pterocarpus erinaceus (Fabaceae) blocked the oestrus cycle of female rats through antigonadotropic activity. Moreover, a study of the plant substances responsible for these effects revealed the presence of phyto-anti-oestrogens in these plant extracts. In order to explain the mechanism by which these substances exert their antifertility actions, the interaction of the plant extract with oestrogen and progesterone receptors was studied. All crude extracts exerted inhibition of ((3)H)-oestradiol or ((3)H)-Organon binding to their respective receptors but their relative affinities were much lower than those of oestradiol or progesterone. Respective efficiencies of plant extracts in competing for the oestrogen receptor were as follows: A. laxiflora > P. erinaceus > C. macrocarpum > C. nitida. The efficiency order of competition for the progestin receptor was different to that of oestrogen. The most potent competitor was C. macrocarpum extract, followed by P. erinaceus, C. nitida and A. laxiflora. Moreover, the interaction between oestradiol and plant extracts with the oestrogen receptor was determined to be competitive only for C. macrocarpum and A. laxiflora, whereas all compounds produced a competitive inhibition on the progestin receptor binding. These results suggest that the plant extract binding site was the same site as for the steroid. These results suggest also that crude plant extracts may interfere with natural oestrogen and/or progestagen in vivo by binding to steroid receptors.

Oestradiol binding to nuclei of anterior pituitary cells of the ram.

The methodology to fully characterise nuclear receptor for oestradiol-17 beta (E2) in the ram pituitary has been investigated. Purified nuclei, clean under the electron microscope, were obtained from 2.4 M sucrose ultracentrifugation and were extracted for 2 h at 0 degrees C with 0.6 M NaCl. After centrifugation, the supernatant was incubated with [3H]E2 with or without a 100-fold excess of unlabelled E2. The main results were: the specific binding was maximum at 20 degrees C in 2-3 h and remained constant up to 19 h without significant metabolism; an incubation temperature of 25 degrees C reduces the binding, while at 0 degrees C maximum binding was attained at a much slower rate; the binding was linearly related to the dose of nuclear proteins; the binding was not affected by DNase and RNase but was suppressed by trypsin, pronase or a temperature of 56 degrees C; binding was specific for oestrogens; preincubation of cytosol with [3H]E2 and then coincubation with nuclei showed an uptake of the [3H]E2 receptor complex by nuclei; such a transfer was inhibited if cytosol was previously heated; after a prelabelled cytosol-nuclei coincubation, a specific binding peak was found in the nuclear extract submitted to sucrose gradient sedimentation (4.1S); in vivo injection of 100 micrograms E2 resulted in a sharp increase in nuclear receptor numbers 30 and 60 min later, with a concomitant drop in cytosolic receptor numbers. These results indicate that E2 can bind to pituitary nuclei in the ram.

Antiestrogen binding within different pituitary cell populations. Comparison with androgen and estrogen receptors.

Gonadotrope-enriched populations were prepared from 42-day old male rats by centrifugal elutriation. They contained 4.8 +/- 0.7% of the cells, 51 +/- 10% of the LH and less than 3% of the PRL (n = 4). Gonadotrope-depleted fractions were also obtained that contained most of PRL cells. Specific antiestrogen binding sites (AEBS) were quantitated in these populations after destruction of estrogen receptor. Results showed the presence of a distinct, specific high affinity binding site for antiestrogen in dispersed pituitary cells and in enriched fractions. However, AEBS are not specific of a pituitary cell type. Thus, AEBS appear different from estrogen receptors in pituitary gland: by the thermal stability of AEBS, by the localization of AEBS in particulate material, by the uniform distribution of AEBS in different populations which differ markedly for E2 binding sites. Whereas the ratio of binding AE/E2 averaged 11.4 in the initial cell suspension it reached only 2.9 in the gonadotropes. The dissociation constants for AEBS were in the same range (1.16 - 2.27 X 10(-9) M) for the different populations.

Effects of some traditional plant extracts on rat oestrous cycle compared with Clomid.

It is well known that the plant kingdom contains numerous bioactive substances affecting the regulation of reproduction. The present study was undertaken to examine the putative contraceptive effects of three traditional plant extracts from Côte d'Ivoire Pharmacopea. It concerns Afrormosia laxiflora (Papilionacea), Pterocarpus erinaceus (Papilionacea) and Cola nitida (Sterculiacea) stem bark. Data showed that treatment of rats with these plant extracts induced ovulation and oestrous cycle blockade at the dioestrous II stage. The analysis of the principal hormones involved in oestrous cycle regulation showed that the plant extracts decreased gonadotropin release (both LH and FSH). In fact, A. laxiflora, P. erinaceus and C. nitida extracts inhibited gonadotropin release as an antiestrogen-like substance.